During its natural lifecycle, the Lyme disease spirochete, Borrelia burgdorferi, occupies both an arthropod tick vector and a mammalian host. To persist in these two very distinct environments, the bacterium undergoes significant adaptive changes, which includes altering the expression of several major outer surface (lipo)proteins. This adaptive response is controlled by an enhancer-binding protein (Rrp2) that is responsible for activating an alternative sigma factor cascade, in which one sigma factor (RpoN, sigmaN, CN, C54) modulates the expression of a second alternative sigma factor (RpoS, sigmaS, CS, C38). RpoS expression then activates the transcription of several major outer surface (lipo)proteins. Because some of these rpoS-regulated outer surface (lipo)proteins contribute to the virulence of B. burgdorferi, it is not surprising that mutation of rpoS in B. burgdorferi renders the bacterium non-infectious. These findings underline the importance of studying B. burgdorferi RpoS-mediated gene regulation. While previous microarray experiments have furthered our understanding of global gene regulation by the Rrp2/RpoN/RpoS pathway in B. burgdorferi, the experimental design of comparing gene expression patterns between wild type and mutant strains is not ideal. The optimal experimental approach to identify genes that respond to a particular regulator would be to expose the bacteria to the specific condition(s) that activate the pathway and then observe the resulting changes in gene expression. Unfortunately, the conditions that optimally induce the Rrp2/RpoN/RpoS pathway are exceedingly complex. Therefore, we propose to use an alternative approach in which we artificially induce the regulator of interest (e.g. RpoS) and then assess its impact on gene regulation. In Specific Aim 1, we propose to use our newly developed lac inducible expression system to express RpoS and assess its impact on global gene expression in B. burgdorferi using transcriptional microarrays. This approach using the inducible expression system will allow us to identify those genes that are directly and immediately impacted by RpoS and assess whether there is temporal variation in RpoS-dependent gene activation. The information garnered from these studies will further our understanding of RpoS-mediated regulation, and allow us to make informed decisions regarding RpoS-regulated genes that will be the focus of Specific Aim 3. To date, all large-scale comparative gene expression studies investigating the regulatory role of the Rrp2/RpoN/RpoS pathway in B. burgdorferi gene expression have utilized transcriptional microarray techniques. To augment our transcriptional microarray analyses (Specific Aim 1), in Specific Aim 2, we will use two-dimensional difference in-gel electrophoresis (2D-DIGE) to observe changes in the B. burgdorferi proteome following expression of RpoS from our lac inducible expression system, as well as compare protein profiles between wild type Bb and an isogenic rpoS mutant. This will mark the first time that such a broad proteomic assessment has been carried out in B. burgdorferi specifically addressing the impact of the RpoS pathway on protein expression. We anticipate that the combined results from the transcriptional microarray comparison and the 2D-DIGE proteomic analyses will help us definitively identify and prioritize RpoS-regulated genes for the targeted mutational and phenotypic analyses described in Specific Aim 3. Many questions still exist regarding the full extent of RpoS-dependent global regulation, and it is likely that a number of the genes regulated by rpoS are required for the transmissibility, infectivity, and pathogenicity of B. burgdorferi. However, the majority of genes that are under the control of rpoS have no known predicted function. To begin ascribing functions for these RpoS-regulated genes, in Specific Aim 3, we will use targeted mutagenesis to create B. burgdorferi mutants in individual rpoS-regulated genes and then assess the phenotypes of these mutants in the experimental infectious lifecycle of B. burgdorferi. Although the majority of genes to be studied in this Aim will be identified using data from the transcriptional and proteomic studies in Specific Aims 1 and 2, we have used results from our previous transcriptional microarray comparisons to identify two rpoS- regulated genes which we can characterize as we are pursuing the analyses in Specific Aims 1 and 2. The knowledge gained from the Aims described in this proposal will serve to further our understanding of the regulatory events that modulate B. burgdorferi gene expression. We also will be able to begin ascribing functions for individual RpoS-regulated genes in the infectious lifecycle of B. burgdorferi, which will help us identify potential therapeutic targets that can be used to prevent and/or treat Lyme disease.